High-Resolution Mapping of Bacterial Transcriptional Responses in Human-Associated Microbiota

人类相关微生物群中细菌转录反应的高分辨率图谱

• 批准号: 10710183
• 负责人: Ilana Lauren Brito
• 金额: $ 32.05万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-26 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10710183
• 关键词: Activities of Daily Living; Acute; Address; Adopted; Antibiotic Resistance; Antibiotics; Antibodies; Bacteria; Bacterial Genome; Base Pairing; Benchmarking; Cells; Chemistry; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Colorectal Cancer; Communities; Complex; Cytolysis; DNA Damage; DNA sequencing; DNA-Directed RNA Polymerase; Data; Data Set; Development; Diet; Disease; Environment; Escherichia coli; Eukaryota; Excision; Exercise; Fusobacterium nucleatum; Gene Expression Profile; Genetic Transcription; Genus Vanilla; Goals; Growth; Health; Heat-Shock Response; Homeostasis; Human; Human Microbiome; Immunoprecipitation; Inflammatory; Inflammatory Bowel Diseases; Laboratories; Laboratory culture; Libraries; Maps; Metabolic; Metagenomics; Methods; Microbe; Modification; Noise; Operon; Organism; Output; Oxygen; Pathway interactions; Periodontal Diseases; Polymers; Positioning Attribute; Prokaryotic Cells; Protocols documentation; Proxy; RNA; RNA Decay; RNA Stability; Reaction; Regulation; Research Personnel; Resolution; Ribosomal RNA; Role; Running; Sampling; Shotgun Sequencing; Signal Transduction; Site; Stimulus; Stress; Structure; Taxonomy; Technology; Testing; Time; Transcript; Transcription Elongation; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Untranslated RNA; Virulence Factors; Xenobiotics; bacterial community; biological adaptation to stress; clinically relevant; computational suite; computerized tools; cost; cost effective; diverse data; environmental stressor; experimental study; feasibility testing; genetic manipulation; human microbiota; improved; insight; metagenomic sequencing; microbial; microbial community; microbiome; microbiome sequencing; pathogen; polymerization; posttranscriptional; quorum sensing; resilience; resistance gene; response; stressor; success; tool; transcriptome sequencing; transcriptomics; tripolyphosphate

Project Summary Functional profiling of microbial communities is critical to understanding their overall effects on host health. Most often, metagenomic shotgun sequencing of microbiome samples is used to assess total functional capacity. Yet, transcriptional responses may vary dramatically between organisms depending on the context, with potentially large effects. Many metabolic functions are only expressed after the organism acutely senses the presence of particular substrates in their environment. Pathogens may only express virulence factors after obtaining a critical quorum of pathogens. Overall, stress responses are critical for survival under changing abiotic and biotic conditions. Being able to comprehensively map out these pathways, which determine the resilience, plasticity, and patho-functions of the microbiome, requires sensitive, robust transcriptional –omics tools. Performing traditional RNAseq analyses on bacterial communities has been the predominant method to gain transcriptional information, but it is hampered by the need for technical workarounds and it provides incomplete information about the transcriptional landscape. Ribosomal RNA needs to be depleted prior to sequencing, it has a poor signal-to-noise ratio arising from varying RNA decay rates, and it is insensitive to the transcription of non-coding RNA that has secondary structure or post-transcriptional modifications. Alternatively, the position of RNA polymerase (RNAP) can be assessed, which provides a real-time readout of transcription. Although so-called nascent transcript sequencing has been performed in E. coli, revealing transcriptional pause sites and other phenomenon elusive when using RNAseq alone, these protocols rely on immunoprecipitation of RNAP and are therefore unsuitable for complex microbial communities where RNAP may be quite diverse and require species-specific antibodies. As a solution, Precision Run-On and SEQuencing (PRO-seq), a method originally created for examining transcription in eukaryotes, may provide an unbiased method to examine transcriptional dynamics on cultured bacteria or in complex microbial communities, such as the human microbiome. Our goal is to test the feasibility of PRO-seq when applied to prokaryotes and to evaluate its ability to capture transcriptional dynamics associated with canonical stress response pathways (heat-shock, oxygen exposure and DNA damage), using a set of quantitative metrics. We aim to validate, and if necessary, modify the protocol so it can be used robustly across species. We plan to develop a computational approach to test the full breadth of transcriptional phenomena that can be observed using this method, such as transcriptional pausing, bidirectional transcription, differences in RNAP function apparent across species, and RNA decay rates, among other aspects. If successful, we expect that PRO-seq will be adopted to study the responses of human-associated microbiota to host diet, inflammatory signals, xenobiotics and to human transcriptional circuitry, more directly.

项目摘要 微生物群落的功能分析对于理解其对宿主健康的整体影响至关重要。最常见的是 微生物组样品的宏基因组shot弹枪测序用于评估总功能能力。但是，转录 根据上下文，在生物体之间的反应可能会发生巨大变化，并可能具有很大的影响。许多 仅在生物体急性地感觉到特定底物中的存在之后，代谢功能才能表达 环境。病原体只有在获得关键的病原体方面才能表达病毒因子。总体而言，压力 在不断变化的非生物和生物条件下，反应对于生存至关重要。可以全面绘制 这些途径决定了微生物组的弹性，可塑性和病原体，需要敏感， 强大的转录 - 组工具。 对细菌群落进行传统的RNASEQ分析一直是获得的主要方法 转录信息，但由于需要技术解决方案而受到阻碍，并且提供了不完整的 有关转录格局的信息。核糖体RNA需要在测序之前耗尽，其较差 由RNA衰减速率变化而产生的信噪比，对非编码RNA的转录不敏感 具有次要结构或转录后修改。另外，RNA聚合酶（RNAP）的位置 尽管所谓的新生转录本测序具有 在大肠杆菌中进行，揭示单独使用rnaseq时，揭示了转录暂停位点和其他现象难以捉摸， 这些方案依赖于RNAP的免疫沉淀，因此不适合复杂的微生物群落 RNAP可能非常多样化，需要特定规格的抗体。作为解决方案，精密跑步和 测序（Pro-Seq）是一种最初用于检查真核生物中转录的方法，可能会提供无偏见的 检验培养细菌或复杂微生物群落的转录动力学的方法，例如 人类微生物组。 我们的目标是在应用于原核生物时测试Pro-Seq的可行性并评估其捕获的能力 与规范应力响应途径相关的转录动力学（热震，氧气和DNA 损坏），使用一组定量指标。我们的目标是验证，并在必要时修改协议，以便可以使用 跨物种坚固。我们计划开发一种计算方法来测试转录现象的全部广度 可以使用这种方法观察到，例如转录暂停，双向转录，RNAP的差异 跨物种和RNA衰减速率的功能以及其他方面。如果成功，我们希望Pro-Seq将会 被采用以研究与人类相关的微生物对宿主饮食，炎症信号，异种生物和对的反应 人类转录电路，更直接。